Leukocyte integrin aM[unreadable]2 (CD11b/CD18, Mac-1) plays a pivotal role in normal protective inflammatory response and pathological inflammation. This receptor has prodigious adhesive and signaling capabilities which allowed it to become the premier workhorse in host defense. It is also a potential therapeutic target in many diseases in which inflammation plays an essential role, including cardiovascular diseases. The diverse functions and activities ascribed to aM[unreadable]2 arise from its ability to bind a multitude of structurally diverse ligands. However, the mechanisms which allow aM[unreadable]2 to exhibit broad ligand recognition are still poorly understood. Our previous studies with a prototype a aM[unreadable]2 ligand fibrinogen provided initial insight into the mechanism by which the aMI-domain of the receptor recognizes its ligands. In the past funding period we have solved the consensus aMI-domain recognition motif, we termed IRM. A key feature of IRM is a small core consisting of specific combinations of basic and hydrophobic amino acid residues ubiquitous in many aM[unreadable]2 ligands. The characteristics of IRM are consistent with the capacity of aM[unreadable]2 to recognize a wide variety of unrelated sequences and, thus, form a molecular basis for aM[unreadable]2 ligand binding promiscuity. Specific Aim1 is to further characterize the mechanism underlying broad recognition specificity of aM[unreadable]2. Combinatorial peptide libraries and mutational analyses will be used to clarify the structural features of IRM. Mass spectrometry will be used to determine the effect of inflammation-associated protein modifications on the function of IRM. Our preliminary studies revealed that neutrophil secretion products are enriched in IRMs which allowed their prediction as a new class of aM[unreadable]2 ligands. We have found that one of them, human neutrophil cathelicidin peptide LL-37, effectively binds aMb2 and induces a potent aM[unreadable]2- dependent migratory response. Based on this finding we propose that LL-37 and other neutrophil-derived proteins/peptides exert their potent immunomodulatory effects by binding aM[unreadable]2 on monocyte/macrophages. Specific Aim 2 is to test this hypothesis by characterizing aM[unreadable]2-dependent monocyte responses elicited by LL-37. The effect of LL-37 on signaling and migratory functions of aM[unreadable]2 will be determined using aM[unreadable]2- expressing and aM[unreadable]2-deficient cells and in the in vivo animal model. Studies over the past funding period identified integrin aD[unreadable]2 as a multi-ligand receptor with specificity similar to that of aM[unreadable]2 and revealed that its upregulation on inflammatory macrophages inhibits their migration. Specific Aim 3 is to characterize the role of aM[unreadable]2 and aD[unreadable]2, two most abundant and adhesive integrins on macrophages, in emigration of these cells from the inflammatory site during the resolution of inflammation. The efflux of macrophages by draining lymphatics will be investigated in wild-type and integrin-deficient mice. Overall, these studies will lead to an increased understanding of the principles which govern ligand recognition by aM[unreadable]2, will give new insights into the biology of aM[unreadable]2 and aD[unreadable]2 and may be useful in the design of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Inflammation is critically involved in the pathogenesis of many disorders, including cardiovascular disease. Integrin aM[unreadable]2 (Mac-1) is the most versatile receptor on leukocytes and mediates numerous responses of these cells during the inflammatory response. The multiplicity of functions exhibited by Mac-1 depends on its ability to bind a myriad of diverse proteins. Understanding the molecular basis for the extreme stickiness of Mac-1 and the biological significance of receptor's broad recognition could lead to new methods of treatment of disorders in which inflammation plays a role.